Mutations at the murine dominant-white spotting locus (W) (c-Jtir) affect various aspects of hematopoiesis. We have made antibodies against c-Kit with the synthetic peptides deduced from the murine c-kit gene and examined the role of c-Kit in erythropoiesis. The antibody inhibited the stromal cell-dependent large colony formation of the erythroid progenitors. In the culture of erythropoietin-responsive erythroid progenitors of the anemia-inducing Friend virus-infected mouse spleen, the antibody inhibited only proliferation, but not differentiation of the progenitor cells. The inhibition was effective only at the early phase (within 6 hours after erythropoietin addition) before the cells start to proliferate induced by erythropoietin. During the early phase, erythropoietin down-regulated c-kit gene expression. These results suggest a mechanism of combined action of c-Kit with erythropoietin on the lineage-restricted erythroid progenitor cells.

Mutations at the murine dominant-white spotting locus (W) affect various aspects of hematopoiesis. The defect exerted by W mutations is intrinsic to hematopoietic cells and the hematopoietic microenvironment in these mice is unaffected and able to support hematopoiesis. Recently, Chabot et al. (1988) have mapped the protooncogene c-kit to the W locus on mouse chromosome 5 and Geissler et al. (1988) subsequently showed that the c-kit gene is rearranged in two different W mutants. The c-kit gene encodes a transmembrane kinase similar to CSF-1R and PDGFR (Qui et al., 1988; Yarden et al., 1987). A 145 ×103MT tyrosine-specific transmembrane kinase has been identified and characterized in brain tissue extracts and in human glioblastoma cell line (Yarden et al., 1987). Recently, SCF or SI factor has been identified as a candidate for the ligand of the c-Kit protein (receptor) (Zsebo et al., 1990; Martin et al., 1990; Huang et al., 1990; Williams et al., 1990; Flanagan et al., 1990). This is consistent with an earlier conjecture that the W locus encodes a cell-surface receptor that functions in cell-to-cell interactions during development and hematopoiesis (Russell, 1979). Most W mutations cause macrocytic anemia and are known to affect the erythroid progenitors (Gregory and Eaves, 1978; Iscove, 1978; Russell, 1979). Expression of c-kit was found to concur with the sites of active erythropoiesis in yolk sac (Orr-Urtreger et al., 1990), fetal liver, adult bone marrow, and in Friend virus-transformed cell lines (Nocka et al., 1989). In addition, c-kit expression was found to be depressed, presumably because of a reduction in c-Zrit-expressing erythroid cell populations in the fetal liver, as a result of the mutation. Whereas levels of the erythroid progenitor cells (CFU-E) were markedly reduced, normal numbers of the granulocyte-macrophage progenitor cells (CFU-GM) were observed in the W[W fetal liver (Nocka et al.,1989 consistent with earlier observations of only minor effects on granulopoiesis in W mutant mice (Russell, 1979).

Apparently, because of the different microenvironments in fetal liver, adult spleen and bone marrow, W mutations affect erythropoiesis to different degrees at different stages of differentiation in the various hematopoietic organs. Thus, it is necessary to examine the direct role of c-Kit on the erythropoietic progenitor cells in vitro. Recently, we have demonstrated the in vitro reconstruction of a microenvironment adequate for a rapid expansion of erythroid cells in fetal liver using established fetal liver stromal cells (Ohneda et al., 1990) . In this work, we examined the role of c-Kit on the proliferation and differentiation of the late erythroid progenitors from fetal livers on the supporting stromal cell layers and from the anemia-inducing Friend virus-infected mouse spleens.

Preparation of antibody to c-Kit peptides

Two oligopeptides (KIT1; FKTYFNEMVENKKNEW, KIT2; AQVKHNSWHRGDFNYERQE) deduced from the murine c-Azigene sequence (Qui et al., 1988) were synthesized by a peptide synthesizer (Applied Biosystems Inc.). Two rabbits were immunized by subcutaneous injection with 500 μg of the oligopeptides coupled with keyhole limpet hemocyanin emulsified in Freund’s complete adjuvant, followed by additional injections of 500 μg of the oligopeptide coupled with keyhole limpet hemocyanin emulsified in Freund’s incomplete adjuvant after 2, 4 and 6 weeks. The antisera were obtained and evaluated by ELISA 2 weeks after the final booster. IgG was precipitated with 1.34 volumes of saturated ammonium sulphate and dialyzed against 10 mM sodium phosphate buffer (pH 8.0). The dialyzed materials were applied to a DEAE cellulose column and the flow-through fractions were collected. Amounts of IgG in the flow-through fractions were estimated by ELISA.

Immunoprecipitation and autophosphorylation of c-Kit protein

Immune complex kinase assay was performed essentially as described previously (Majumder et al., 1988). Briefly, mouse brain (400 mg) was homogenized in 2 ml of lysis buffer (50 mM Tris, 150 mM NaCl, 20 mM EDTA, 1% Triton X-100,1% deoxycholate, 1% SDS, 1 mM phenylmethylsulfonyl fluoride, 20 pg/ml leupeptin, ImM sodium vanadate at pH 8.4) and centrifuged at 100,000 g for 45 minutes. The supernatant was subjected to wheat-germ-agglutinin chromatography and pretreated with the non-immune serum. The pretreated materials were immunoprecipitated with KIT1 antibodies that were conjugated with protein A sepharose, and incubated with [γ32-P]ATP. The labeled proteins were separated by SDS-PAGE and visualized by autoradiography.

Preparation of hematopoietic progenitor cells and coculture of hematopoietic cells on stromal cell layers

Preparation of hematopoietic progenitor cells and coculture of hemopoietic cells on stromal cell layers were performed as described previously (Yanai et al., 1989). Hematopoietic progenitor cells were prepared from the liver of 13-day embryos after mating of WB-W/+ x WB-W/+ mice. Hemopoietic progenitor cells were cultured on monolayers of FLS5 and MSS62 cell lines in multiwell plates (Falcon 3047, Becton Dickinson), in Iscove’s modified Dulbecco’s medium (IMDM; Gibco), supplemented with 0.4% methylcellulose, 30% heat-inactivated FBS, 1% bovine serum albumin, 100 pM 2-mercaptoethanol and erythropoietin (recombinant human erythropoietin, generously supplied by Kirin-Amgen Co. Ltd.). After 4 days in culture, erythroid colonies were scored by direct staining with benzidine.

Isolation and culture of splenic erythroblasts

Anemia-inducing Friend virus (FVA, provided by Dr H. Amanuma of RIKEN, The Institute of Physical and Chemical Research, Tsukuba, Japan) was injected intravenously into 6-week-old DBA/2 mice. Twenty days after injection, the enlarged spleens were removed and dissected into single cells. To enrich the immature erythroid progenitor cells, the cells were washed in IMDM and were treated with lysing buffer (155 mM NH4C1,10 mM KHCO3, and 1 mM EDTA) in which mature erythroid cells were selectively lysed. After lysing treatment, less than 3% of the spleen cells were benzidine-positive. The enriched erythroid progenitor cells were cultured in IMDM supplemented with 30% heat-inactivated FBS and 0.1 LJ/ml of erythropoietin, according to the method of Koury et al. (1984).

Detection of genotypes of the littermates by RT-PCR method

The genotypes of the littermates obtained by the mating of WB-W/+ x WB-IV/+ were determined by reverse transcrip-tase-polymerase chain reaction (RT-PCR). The primers used were sense primer (1541-1565 in nucleotide number); 5’-GCCTTCTTTA ACTTTGCATTTAAAG-3’, and antisense primer (2656-2637): 3’-CTTGGAGTCGACCGGCATCC-5’ and these primers were kindly supplied by Dr T. Kunisada of Kumamoto University Medical School, Kumamoto, Japan. The amplified DNA product from the W mutant mRNA is shorter than that of the wild-type mRNA as described by Hayashi et al. (1991). For cDNA synthesis, 10 pg of RNAs from brains (C57BL/6J-+/+ mouse and WB-VF/+ mouse) and from whole embryos (littermates of the mating of WB-W/+ x W/+) were incubated with 100 pmol of random primer and 200 U of MMLV reverse transcriptase (BRL) in 20 pl reaction mixture containing 0.55 M Tris-HCl, pH 8.3, 0.075 M KC1, 3 mM MgC12, 10 mM dithiothreitol, 200 mM deoxyribonucleotide triphosphates, and 20 U of RNAase inhibitor (Pharmacia) at 37°C for 60 minutes. The cDNA was amplified by 2.5 units of Taq DNA polymerase and 40 pmol of sense and antisense primers in 20 ml reaction buffer (10 mM Tris-HCl, pH 8.3, 50 mM KC1,1.5 mM MgCl2, 0.01% gelatin and 0.5 U of Perfect Match (Stratagene) for 28 cycles in a program Temperature Control System PC 700 (Astee Inc.). The amplified DNA products were analyzed by 1.4% agarose gel electrophoresis.

Northern blot analysis of c-kit mRNA and SCF mRNA

The erythroid progenitor cells derived from Friend virusinfected mice were cultured in vitro with 0.1 U/ml erythropoietin and the cells were collected at appropriate time intervals (0–12 hours). The cells were homogenized in guanidine isothiocyanate, and RNAs were isolated by centrifugation through CsCl. 4 μg of poly (A)+ RNA, obtained by oligo(dT)-Latex (Oligotex-dT30, Nihon Synthetic Rubber Co.), were separated in 0.7% agarose gel containing formaldehyde, and transferred to nylon membranes. The membranes were prehybridized and hybridized with the nick-translated 5.1 kb c-kit cDNA (provided by Dr A. Singh, Genentech.Inc.). The amount of c-kit mRNA in each sample was assessed by comparison to hybridization with a β -actin probe.

For detection of SCF mRNA in the stromal cells, total RNAs were isolated from two stromal cell lines, FLS cell lines (Ohneda et al., 1990) and MSS cell lines (Yanai et al., 1989). The SCF cDNA probe was prepared by reverse transcriptase-polymerase chain reaction (RT-PCR) from mRNA of FLS5 cells with sense primer (16–36 nucleotide number by Zsebo et al., 1990) 5’-ATGAAGAAGACACAAACTTGG-3’ and antisense primer (598–579) 3’-ACGTCGGTCCGAGG-GAATCCT-5’. The sequence of the product of RT-PCR was confirmed after subcloning.

The large erythroid colony formation of the progenitor cells of fetal liver from W/W mice

To examine the effect of mutation at the W locus on the erythroid progenitor cells, we compared the erythroid colony formation of the progenitors in the liver of normal (+/+), heterozygous (W/+) and mutant (W/W) embryos at 13 days of gestation. The large erythroid colony assay on the stromal cell layers was performed as described previously (Yanai et al., 1989; Ohneda et al.,1990) On the stromal cell layer, large colonies consisting of 200 to 1,000 benzidine-positive cells were formed from the erythroid progenitor cells which are close to CFU-E stage (Ohneda et al., 1990) after 3-4 days in the semisolid medium with erythropoietin. The direct cell-to-cell contact between the erythroid progenitor cells and the stromal cells seemed to induce rapid expansion of mature erythroid cells (Yanai et al., 1991) To determine the involvement of SCF, the ligand of the c-Kit, on the large colony formation, we measured expression of SCF mRNA in the stromal cells (Fig. 1). SCF mRNA was abundantly expressed in the stromal cells with erythropoietic supporting ability, suggesting the possible involvement of SCF in the stromal cell-dependent large colony formation. Fetal livers were obtained from the offspring of mating W/+ x W/+ animals. W/W homozygotes could be distinguished easily from their +/+ or W/+ littermates on the basis of their small embryo size and the pale color of their livers (Nocka et al., 1989), but +/+ and W/+ littermates could not be distinguished. Recently W mutation was shown to be a point mutation in the splice donor site near the membrane spanning domain of c-Kit and the mutant protein is lacking the membrane spanning domains resulting in no expression of c-Kit on the mutant cell surface (Nocka et al., 1990). The reverse transcriptase-polymerase chain reaction (RT-PCR) method can be applied to distinguish the wild- and mutant-type mRNAs (Hayashi et al., 1991). Three genotypes (WfW, W/+ and +/+) were clearly identified by this method (Fig. 2). In one litter, there was one WfW homozygous fetus (embryo number 3 in Fig. 2) that could be assigned both from the RT-PCR method and from the pale colour of the liver. The erythroid progenitors in the W/W fetus did not form the large colonies on the stromal cell layers (Table 1). In the +/+ and W/+ fetuses, the size of colonies formed on the stromal cell layers showed significant differences depending on the genotypes of the fetuses, although the numbers of colonies did not show notable differences (Fig. 3). The average size of the colonies formed from the progenitors of the homozygous (+/+) fetuses was 0.33 ± 0.10 mm2 (among 7 different colonies), whereas that of the heterozygous progenitors was 0.12 ± 0.04 mm2 (among 21 different colonies), thus the colony sizes of the wild-type progenitors are three times larger than those of the heterozygous progenitors.

Table 1.

Effects of -genotypes of individual embryos and anti-c-Kit antibody on the large erythroid colony formation

Effects of -genotypes of individual embryos and anti-c-Kit antibody on the large erythroid colony formation
Effects of -genotypes of individual embryos and anti-c-Kit antibody on the large erythroid colony formation
Fig. 1.

Expression of SCF mRNA in the stromal cells. Total RNAs were prepared from 6 mouse stromal cell lines, MSS31 (lane 1), MSS62 (lane 2), MSS72 (lane 3), FLS5 (lane 4), FLS8 (lane 5), and FLS9 (lane 6). Total RNAs (10 pg) were analyzed by an agarose gel electrophoresis, blotted to a nylon membrane and hybridized with SCF cDNA probe. An arrowhead shows the band corresponding to SCF mRNA. Ethidium bromide staining of the gel is shown below. Migrations of 28S and 18S rRNAs are shown as molecular weight markers.

Fig. 1.

Expression of SCF mRNA in the stromal cells. Total RNAs were prepared from 6 mouse stromal cell lines, MSS31 (lane 1), MSS62 (lane 2), MSS72 (lane 3), FLS5 (lane 4), FLS8 (lane 5), and FLS9 (lane 6). Total RNAs (10 pg) were analyzed by an agarose gel electrophoresis, blotted to a nylon membrane and hybridized with SCF cDNA probe. An arrowhead shows the band corresponding to SCF mRNA. Ethidium bromide staining of the gel is shown below. Migrations of 28S and 18S rRNAs are shown as molecular weight markers.

Fig. 2.

Genotype analysis of individual embryos obtained by mating of W/+ x W/+ mice. The amplified DNA products obtained using RT-PCR from total RNAs of adult brains of C57BL/6J (+/+), WB-W/+ parents (W/+) and 13-day old embryos of 6 littermates (1-6 obtained by the mating of WB-W/+ x WB-W/+) were separated by 1.4% agarose gel electrophoresis. The sizes of nucleotides in base pairs are indicated at the left. The upper bands were generated from the wild-type mRNA and the lower bands were generated by the mRNA with W mutation.

Fig. 2.

Genotype analysis of individual embryos obtained by mating of W/+ x W/+ mice. The amplified DNA products obtained using RT-PCR from total RNAs of adult brains of C57BL/6J (+/+), WB-W/+ parents (W/+) and 13-day old embryos of 6 littermates (1-6 obtained by the mating of WB-W/+ x WB-W/+) were separated by 1.4% agarose gel electrophoresis. The sizes of nucleotides in base pairs are indicated at the left. The upper bands were generated from the wild-type mRNA and the lower bands were generated by the mRNA with W mutation.

Fig. 3.

Erythroid colony formation on the stromal cell layers. Fetal liver erythroid progenitor cells were obtained from the 13-day embryos of three genotypes (+/+, W/+ and W/W) and cultured on the FLS5 cell layers in a semisolid medium in the presence of erythropoietin (0.1 U/ml). After 4 days incubation, three types of colonies developed from the erythroid progenitor cells; (A) A colony from a hétérozygotie embryo (embryo number 1 in Fig. 2), (B) a colony from a WfW mutant homozygotic embryo (embryo number 3 in Fig. 2), (C) a colony from a wild-type embryo (embryo number 6 in Fig. 2). Bar, indicates 100 μm.

Fig. 3.

Erythroid colony formation on the stromal cell layers. Fetal liver erythroid progenitor cells were obtained from the 13-day embryos of three genotypes (+/+, W/+ and W/W) and cultured on the FLS5 cell layers in a semisolid medium in the presence of erythropoietin (0.1 U/ml). After 4 days incubation, three types of colonies developed from the erythroid progenitor cells; (A) A colony from a hétérozygotie embryo (embryo number 1 in Fig. 2), (B) a colony from a WfW mutant homozygotic embryo (embryo number 3 in Fig. 2), (C) a colony from a wild-type embryo (embryo number 6 in Fig. 2). Bar, indicates 100 μm.

Effect of antibodies against c-Kit on erythropoiesis

To determine the involvement of c-Kit with the erythroid progenitors, anti-c-Kit antibodies were made against two synthetic polypeptides (KIT1; amino acid residues 68–83 and KIT2; 258–276) located in the extracellular domains of the c-Kit polypeptide, deduced from the murine gene (Qui et al., 1988). The antibodies were raised against the KIT1 and KIT2 peptides in the rabbits and the purified IgG of the antibody against the KTT1 peptide (called KIT1 antibody) was used through-out experiments. KIT1 antibody was shown in vitro to immunoprecipitate specifically autophosphorylated c-Kit (ppl65kD) from mouse brain tissue (Fig. 4). In addition, KIT1 antibody exhibited specific immuno-staining of the erythroid progenitors of fetal fiver by FACS analysis (unpublished observation).

Fig. 4.

Autophosphorylation of immunoprecipitated c-Kit brain extracts purified by wheat germ agglutinin affinity chromatography were pretreated with preimmune serum and were precipitated with KITl antibody. Then, the precipitates were mixed with protein A-sepharose and the protein A-immunocomplexes were incubated with [β-32P] ATP. The autophosphorylated proteins were analyzed by SDS-PAGE and the protein specifically autophosphorylated in KIT1 antibody is indicated by an arrow. Mr markers are indicated at left. Lane 1: preimmune serum, lane 2: KITl antibody.

Fig. 4.

Autophosphorylation of immunoprecipitated c-Kit brain extracts purified by wheat germ agglutinin affinity chromatography were pretreated with preimmune serum and were precipitated with KITl antibody. Then, the precipitates were mixed with protein A-sepharose and the protein A-immunocomplexes were incubated with [β-32P] ATP. The autophosphorylated proteins were analyzed by SDS-PAGE and the protein specifically autophosphorylated in KIT1 antibody is indicated by an arrow. Mr markers are indicated at left. Lane 1: preimmune serum, lane 2: KITl antibody.

Antibodies were added to culture media for the large erythroid colony assay on the stromal cell layers. KIT1 antibody considerably inhibited the formation of large erythroid colonies, whereas normal rabbit IgG showed essentially no inhibition (Table 1). KIT2 antibody also gave essentially the same inhibition (data not shown).

These results indicate that the antibodies against the c-Kit interfere with the c-Kit function in the erythroid colony formation of the hematopoietic progenitor cells of fetal liver.

Effect of KITl antibody on the erythroid cells of anemia-inducing Friend virus-infected mice

In the spleen of mice infected with anemia-inducing strain of Friend virus complex, erythroid progenitor cells accumulated close to BFU-E (Peschle et al., 1980). The proliferation and differentiation of these erythroid progenitor cells can be induced with erythropoietin in the culture (Koury et al., 1984). The progenitor cells of spleens, 20 days after infection with Friend virus complex, were obtained after the lysing procedure and cultured in the presence of 0,1 U/ml erythropoietin (Fig. 5A). An eight-fold increase in the cell number was observed after a lag phase of 1 day and the cell number decreased after 3 days in culture. This in vitro culture required a high concentration (30%) of fetal bovine serum, but at a concentration less than 30%, the differentiation and proliferation of the progenitor cells was greatly reduced. The proportion of the benzidine-positive, hemoglobin-producing cells increased from 1-2% to 70% after 2 days culture in the presence of erythropoietin. In the culture without erythropoietin, the cells gradually decreased in number because of the programmed cell death (Koury and Bondurant, 1990) and the benzidine-positive cells were not observed. Thus, the differentiation and proliferation, in the in vitro culture, of the erythroid progenitor cells are strictly erythropoietin-dependent. When KIT1 anti-body was added to the culture, the proliferation of the progenitor cells was inhibited. Inhibition with the antisera was dose-dependent but the highest inhibition was 40% (Fig. 5B).

Fig. 5.

Effect of KIT1 antibody on the proliferation of erythroid progenitor cells derived from Friend virus-infected spleens. (A) The erythroid progenitor cells of Friend virus-infected spleens were cultured at lxlO5 cells/ml with 0.1 U/ml erythropoietin (open circles) containing KTT1 antibody (0.1 mg/ml) (closed triangles) or normal rabbit IgG (0.1 mg/ml) (closed circles). Similarly, the cells were cultured in the absence of erythropoietin with (closed squares) or without normal rabbit IgG (open squares). The living cells were counted using a hemocytometer at 24 hour intervals. (B) To test the dose-dependent effect of KIT1 antibody on the proliferation of the erythroid progenitor cells, various concentrations of KIT1 antibody were added and the cell number was counted using a hemocytometer. Each point represents the mean of two samples.

Fig. 5.

Effect of KIT1 antibody on the proliferation of erythroid progenitor cells derived from Friend virus-infected spleens. (A) The erythroid progenitor cells of Friend virus-infected spleens were cultured at lxlO5 cells/ml with 0.1 U/ml erythropoietin (open circles) containing KTT1 antibody (0.1 mg/ml) (closed triangles) or normal rabbit IgG (0.1 mg/ml) (closed circles). Similarly, the cells were cultured in the absence of erythropoietin with (closed squares) or without normal rabbit IgG (open squares). The living cells were counted using a hemocytometer at 24 hour intervals. (B) To test the dose-dependent effect of KIT1 antibody on the proliferation of the erythroid progenitor cells, various concentrations of KIT1 antibody were added and the cell number was counted using a hemocytometer. Each point represents the mean of two samples.

To investigate the timing of c-Kit function, after erythropoietin addition on the erythroid progenitor cells, KIT1 antibody was added to the culture at different times after erythropoietin addition. KIT1 antibody only inhibited the proliferation within 6 hours after the addition of erythropoietin, when the cell division had not yet started (Fig. 6A). Conversely, monoclonal antibody TER-119, which is specific to erythroid cells (Y. Kina, personal communication), showed only slight inhibition in a time-independent fashion. Thus, c-Kit may function only at the very early period of the progenitor cells’ response to erythropoietin action. Interestingly, with the addition of KITl antibody, there was no reduction in the proportion of the hemoglobin-positive cells (Fig. 6B). Thus, KITl inhibited proliferation, but not differentiation of the erythroid progenitor cells at the very early period of the erythropoietin-mediated cellular signaling process.

Fig. 6.

Time-dependent effects of K1T1 antibody on the proliferation and differentiation of the erythroid progenitor cells. (A) Effect of K1T1 antibody on the cell proliferation. At appropriate times (0,3,4,5,6,12 and 24 hours) after the culture of the erythroid progenitor cells, KIT1 antibody (0.1 mg/ml) was added to the culture (closed circles). After 48 hours, cells were collected and their numbers estimated. Monoclonal antibody specific to erythroid cells termed TER-119 was used as a control (open circles). (B) Effect of antisera on differentiation. The percentage of the benzidine-positive cells is shown as the columns and the presence or absence of erythropoietin (Epo), KIT1 antibody (KIT1 Ab) and normal rabbit IgG (rabbit IgG) is shown as + or — below the columns. Preculture time shows hours before the addition of KIT1 antibody to the culture.

Fig. 6.

Time-dependent effects of K1T1 antibody on the proliferation and differentiation of the erythroid progenitor cells. (A) Effect of K1T1 antibody on the cell proliferation. At appropriate times (0,3,4,5,6,12 and 24 hours) after the culture of the erythroid progenitor cells, KIT1 antibody (0.1 mg/ml) was added to the culture (closed circles). After 48 hours, cells were collected and their numbers estimated. Monoclonal antibody specific to erythroid cells termed TER-119 was used as a control (open circles). (B) Effect of antisera on differentiation. The percentage of the benzidine-positive cells is shown as the columns and the presence or absence of erythropoietin (Epo), KIT1 antibody (KIT1 Ab) and normal rabbit IgG (rabbit IgG) is shown as + or — below the columns. Preculture time shows hours before the addition of KIT1 antibody to the culture.

Erythropoietin down-regulates c-kit gene expression in the erythroid cells

The time-dependent inhibition of proliferation of the erythroid progenitor cells with KITl antibody suggested the involvement of c-Kit in the early event induced by the erythropoietin and also a close fink between c-Kit and erythropoietin receptor actions. It has been reported that c-kit mRNA was detectable in the fetal liver cells and in some murine erythroleukemic cells (Nocka et al., 1989). We examined whether erythropoietin mimics c-kit gene expression, in the in vitro culture of erythroid progenitor cells, by northern blot hybridization. Measurement of c-kit mRNA levels after the induction of the progenitor cells with erythropoietin indicated that erythropoietin down-regulated expression of the c-kit gene soon after the erythropoietin addition (Fig. 7) and thus, the involvement of c-Kit in the proliferative response of the erythroid progenitors is rapid and transient. This is consistent with the results obtained by the inhibition with KITl antibody.

Fig. 7.

Expression of c-kit mRNA in the erythroid progenitor cells. After appropriate times (0, 3, 6, 12 hours) of culture with erythropoietin, the erythroid progenitor cells derived from Friend virus-infected spleens were collected and poly (A)+ RNA was isolated. Four micrograms of mRNA in each sample was loaded on agarose gel and the hybridization was performed as described in Materials and methods. Messenger RNA from adult brain was used as a positive control and levels of β- actin mRNA were titrated for the equalization in each sample. An arrowhead shows the band corresponding to c-kit mRNA.

Fig. 7.

Expression of c-kit mRNA in the erythroid progenitor cells. After appropriate times (0, 3, 6, 12 hours) of culture with erythropoietin, the erythroid progenitor cells derived from Friend virus-infected spleens were collected and poly (A)+ RNA was isolated. Four micrograms of mRNA in each sample was loaded on agarose gel and the hybridization was performed as described in Materials and methods. Messenger RNA from adult brain was used as a positive control and levels of β- actin mRNA were titrated for the equalization in each sample. An arrowhead shows the band corresponding to c-kit mRNA.

c-Kit (gene product of W; murine dominant white-spotting locus) is important in the growth and differentiation of hematopoietic cells, but its mechanism of action is not known. W mutation in mice affected erythropoiesis as well as the very early stem cells (McCulloch et al., 1964; Gregory and Eaves, 1978). In vitro colony formation of the erythroid progenitor cells in fetal livers of the mutant mice suggested that c-Kit may have a function at the CFU-E stage, but not at the BFU-E stage (Nocka et al., 1989). However, it is not clear how c-Kit regulates the proliferation and differentiation of these erythroid progenitor cells.

To examine the action of c-Kit on the erythroid progenitor cells, we used a new assay method for the erythroid progenitors using the large erythroid colony formation supported by the stromal cells established from the mouse erythropoietic organs (Yanai et al., 1989; Ohneda et al., 1990). The large colony formation on the stromal cell layers requires erythropoietin and intimate direct cell-to-cell contact. Many mature eryth-rocytes and erythroblasts showing cytoplasmic budding were produced on the stromal cell layers (Yanai et al.,1991) This in vitro system supports more than 10 cycles of cell division of the erythroid progenitor cells (Ohneda et al., 1990) which is sufficient to explain the phase of rapid expansion of the erythropoietic cell population observed in the fetal liver (Wolf and Trentin, 1967).

Using this assay, we demonstrated that colonies were not formed from fetal liver erythroid progenitors of the W/W homozygous embryos and the sizes of the colonies of the erythroid progenitors in the heterozygous (W/+) embryos were significantly reduced compared to those in the wild-type (+/+) littermates (Fig. 3). The stromal cells used in this work were shown to express SCF mRNA and thus the stromal cell-dependent large colony formation may require SCF.

Mutation in W homozygous mice is identified as a single base substitution at the 5’-splice donor site of the exon which encodes the transmembrane domain (Nocka et al., 1990; Hayashi et al., 1991) and the mutated c-Kit molecules may be lost from the cell surface because of the loss of the transmembrane domain caused by the aberrant exon skipping (Nocka et al., 1990). Thus, the amount of the c-Kit molecules per cell in the heterozygous (W/+) progenitor cells may be half of that in the wild-type (+/+) cells. The reduction in the cell number in the colonies derived from the heterozygous erythroid progenitor cells seems to corre-late well with the reduction in the c-Kit molecules on the progenitor cells. However, no significant difference was observed in the number of the colonies formed from the W/+ and +/+ progenitor cells. c-Kit may affect the erythropoietin-reactive late erythroid pro-genitor cells more than the earlier progenitor cells, as suggested by Nocka et al. (1989). The quantitative difference indicates that c-Kit levels determine the number of the cell division cycles thus affecting the extent of expansion of the erythroid progenitors.

The results obtained using anti-c-Kit antibody in two different assay systems, including the large erythroid colony formation on the stromal cells and in vitro culture of the Friend virus-infected spleen erythroblasts indicated that c-Kit is required for proliferation of the erythroid progenitor cells, but not for differentiation.

FVA-infected erythroid progenitors are arrested at the erythropoietin responsive stage (possibly at the BFU-E to CFU-E stage) and can be induced to proliferate and differentiate in vitro with the addition of erythropoietin in liquid culture containing a high concentration of fetal bovine serum (Koury et al., 1984). It is possible that the soluble form of SCF in the serum may be required for the differentiation and proliferation of FVA-infected erythroid progenitor cells. In fact, the soluble form of SCF was shown to be active in in vivo and in vitro hematopoiesis (Matin et al., 1990). Erythropoietin induces cell division after a lag of 1 day, which may be required for the activation of the mitogenic signaling pathways inherent to these cells. Anti-c-Kit antibody inhibited the proliferation, but not the differentiation of the FVA-infected eryth-roid progenitors.

Incomplete inhibition of the proliferation of the erythroid progenitor cells by the anti-c-Kit antibody (Fig. 6A) suggests the presence of different cell populations among the Friend virus-arrested erythroid progenitor cells. One cell population may be committed to the mitogenic program and the anti-c-Kit antibody cannot affect such a cell population. The other population may not be committed and the action of c-Kit is required for proliferation to be induced by erythropoietin addition. In either cell population, differentiation can be induced by erythropoietin, thus, the proliferative signals and differentiation signals induced by erythropoietin may be separable as suggested previously (Noguchi et al., 1988). SCF may act as a “competence” factor and erythropoietin may act as a “progression” factor. This is similar to the action of PDGF as a “competence” or “early response” factor (Stiles et al., 1979) and this functional similarity is plausible because PDGF-receptor and c-Kit have a strong structural similarity (Qui et al., 1988). Since c-Kit has tyrosine kinase activity in its cytoplasmic domain and erythropoietin receptor has no such domain (D’Andrea et al., 1989), it is interesting to know how erythropoietin drives the c-Kit-mediated mitogenic signals in the erythroid progenitor cells. This requires the analysis of combinatorial mechanisms between the erythropoietin receptor and the c-Kit molecules as has been demonstrated in the erythropoietin receptor and Friend virus envelope (gp55) (Li et al., 1990).

The growth inhibition by the anti-c-Kit antibody was only observed within the first 6 hours after addition of erythropoietin. This suggests that c-Kit acts during the very short period required for the activation of the mitogenic signaling pathways induced by erythropoietin. This event is sufficient to induce 3 successive cycles of cell division of the erythroid progenitor cells. This is consistent with the result that erythropoietin down-regulates c-kit gene expression shortly after erythropoietin addition. The down-regulation may be required for the cycling progenitor cells to switch to the differentiation and maturation program. Since 10 cycles of cell divisions occur in the erythroid progenitor cells on the stromal cell layers (Ohneda et al., 1990), the extent of the expansion of the erythroid progenitor cells may be determined by the level of c-Kit molecules in the cells and/or SCF.

We propose a model for the control of differentiation and proliferation of the erythroid progenitor cells as follows: c-Kit may act on growth expansion both of the very early cycling hematopoietic stem cells and of some lineage-restricted progenitors such as erythroid cells. The progenitors committed to a specific lineage cannot proliferate unless the lineage-specific factor exists. The lineage-specific factor may mimic signal transduction through c-Kit and only progenitor cells exhibiting c-Kit function can be induced by the lineage-specific factors such as erythropoietin. The stromal cells with the surface-attached SCF molecules may locate in the particular organ and provide a microenvironment suitable for the expansion of the specific lineage of the progenitors. In fact we and others have found that specific stromal cells can selectively stimulate the specific lineage of the progenitors (Yanai et al., 1989; Ohneda et al., 1990; Zipori et al., 1985). Whereas the phenotypes of these mutations are restricted to hematopoiesis, melanogenesis and spermatogenesis (Russell, 1979), the expression of c-kit (Orr-Urtreger et al., 1990) and SCF mRNAs (Matsui et al., 1990) are detected in a variety of mouse organs. Thus, the multiple, but cell-lineage restricted effects of the W/W and Sl/Sl mice can be explained by these kinds of combined action between the lineage-specific factors and c-Kit-SCF. It is of interest to investigate similar combined action between c-Kit and the lineage-specific factors in other progenitor cells.

This work was partly supported by Grant-in-Aid for Cancer Research from the Ministry of Education, Science and Culture of Japan and Special Coordination Funds from the Science and Technology Agency of the Japanese Government. Anemia strain of Friend virus stock was kindly supplied by Dr H. Amanuma of Tsukuba Life Science Center, The Institute of Physical and Chemical Research, Tsukuba. Thanks are due to Drs T. Kunisada and S.-I. Nishikawa of Kumamoto University School of Medicine, Kumamoto, for providing us with the oligonucleotides for PCR to detect c-kit mutation(s) and for helpful suggestions for these techniques. The oligopeptides deduced from c-kit gene product were kindly synthesized by Dr S. Abe of Research Institute of Nihon Kogyo Co. Saitama. We thank Dr A. Singh of Genentech Inc. California for providing human c-kit cDNA clone and Dr Y. Kina of Research Institute for Chest Disease, Kyoto University, Kyoto for providing monoclonal antibody TER-119 specific to erythroid cells.

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